International Journal of Pharmaceutics 510 (2016) 493–500

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International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical nanotechnology

Microfluidic assisted synthesis of silver nanoparticle–chitosan

composite microparticles for antibacterial applications
Chih-Hui Yanga , Lung-Shuo Wangb,c, Szu-Yu Chena,b , Mao-Chen Huanga,b , Ya-Hua Lia,b ,
Yun-Chul Lina,b , Pei-Fan Chena,b , Jei-Fu Shawa,* , Keng-Shiang Huangb,*
a
Department of Biological Science and Technology, I-Shou University, Taiwan
b
The School of Chinese Medicine for Post-Baccalaureate, I-Shou University, Taiwan
c
Department of Chinese Medicine, E-Da Hospital, Kaohsiung, Taiwan

A R T I C L E I N F O A B S T R A C T

Article history: Silver nanoparticle (Ag NP)-loaded chitosan composites have numerous biomedical applications;
Received 22 September 2015 however, fabricating uniform composite microparticles remains challenging. This paper presents a novel
Received in revised form 16 December 2015 microfluidic approach for single-step and in situ synthesis of Ag NP-loaded chitosan microparticles. This
Accepted 5 January 2016
proposed approach enables obtaining uniform and monodisperse Ag NP-loaded chitosan microparticles
Available online 15 January 2016
measuring several hundred micrometers. In addition, the diameter of the composites can be tuned by
adjusting the flow on the microfluidic chip. The composite particles containing Ag NPs were
Keywords:
characterized using UV–vis spectra and scanning electron microscopy-energy dispersive X-ray
Silver
Nanoparticles
spectrometry data. The characteristic peaks of Ag NPs in the UV–vis spectra and the element mapping
Chitosan or pattern revealed the formation of nanosized silver particles. The results of antibacterial tests indicated
Microfluidic that both chitosan and composite particles showed antibacterial ability, and Ag NPs could enhance the
Synthesis inhibition rate and exhibited dose-dependent antibacterial ability. Because of the properties of Ag NPs
Antibacterial and chitosan, the synthesized composite microparticles can be used in several future potential
applications, such as bactericidal agents for water disinfection, antipathogens, and surface plasma
resonance enhancers.
ã 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).

1. Introduction loaded chitosan composites exhibit remarkably strong antimicro-

Composite materials are composed of two or more constituent
materials. Substantial attention has been devoted to combining
different functional materials into composite materials for
multifunction applications (Zhang et al., 2015a). Furthermore,
the characteristics of composite materials may be more favorable
than those of the sum of the individual components (Pelgrift and
Friedman, 2013). Therefore, composite materials have a substantial
effect across a wide range of fields (Esther et al., 2015; Sacco et al.,
2015; Zhang et al., 2015b; Wang et al., 2015a; Gogoi and
Chowdhury, 2014; Li et al., 2013; González-Campos et al., 2013;
Han et al., 2013). Various methods for synthesizing composite
materials, particularly with functional polymers, were proposed
(Patrícia et al., 2015). Silver nanoparticle (Ag NP)-loaded chitosan
composites have recently been of interest for numerous biomedi-
cal applications (Alexander et al., 2015; Reidy et al., 2013). Ag NP-
* Corresponding authors.
E-mail addresses: shawjf@isu.edu.tw (J.-F. Shaw), huangks@isu.edu.tw
(K.-S. Huang).
bial activity against gram negative or gram positive bacteria
(Annur et al., 2015; Ito et al., 2015; Ishihara et al., 2015; Liu et al.,
2015; Anisha et al., 2013; Fouda et al., 2013; Regiel et al., 2013).
However, Ag NPs have high surface energies and tend to aggregate
and fuse, resulting in processing, storage, and application
difficulties (Alexandru et al., 2013). Encapsulating Ag NPs in
matrix systems could provide an efficient approach for preventing
aggregation (Sobiya et al., 2014). In addition, chitosan polymer is a
remarkable antimicrobial agent for inhibiting bacterial or fungal
growth. Combining Ag NPs with chitosan polymer can provide
more potent and diverse antimicrobial activity (Wang et al.,
2015b).
Various methods for fabricating Ag NP-loaded chitosan
composites have been developed. For example, Zain et al. (2014)
used ascorbic acid reduction and microwave heating for preparing
Ag NP-loaded chitosan composites, and the results revealed that
the particle size can be increased by either increasing the nitrate
concentration or reducing the chitosan concentration. Hassabo
et al. (2015) used glucose as a reducing agent and impregnated
various polysaccharide substrates with Ag NPs measuring

http://dx.doi.org/10.1016/j.ijpharm.2016.01.010
0378-5173/ã 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
494 C.-H. Yang et al. / International Journal of Pharmaceutics 510 (2016) 493–500

Fig. 1. Schematics of the synthesis of Ag NP-loaded chitosan composite microparticles (not to scale). Emulsions were formed at the cross-junction of the microfluidic chip and
subsequently dropped into a NaOH solution for silver nanoparticle reduction and chitosan solidification. The diameter of emulsions was controlled by adjusting the flow
conditions. The inset is a photograph of the middle chip. The numbers 1 and 2 indicate inlets; 3 indicates the cross-junction design; 4 indicates the broaden channel design; 5
indicates the outlet, and 6 indicates the screw orifices.

3.7–5.6 nm. Luo et al. (2014) used the Tollens reaction and one-pot
reaction for Ag NPs synthesis; quaternized chitosan and rectorite
were the reducing and stabilizing agents, respectively. Lee et al.
(2014) utilized an electrospinning technique for chitosan nano-
fibers containing various ratios of Ag NPs. Mohamed and Sabaa
(2014) synthesized carboxymethyl chitosan–Ag NP hydrogels
measuring 9–16 nm through an in situ preparation reaction that
involved cross-linking carboxymethyl chitosan with epichlorohy-
drin in an alkaline medium containing silver nitrate. Hebeish et al.
(2014) utilized in situ chemical reduction of Ag ions in the graft
copolymerization of acrylonitrile on chitosan. Lavorgna et al.
(2014) used silver ions instead of Na(+) ions in a reaction for
synthesizing silver–montmorillonite nanoparticles and subse-
quently mixed silver–montmorillonite NPs with chitosan to form
composites. Liu et al. (2013) used a modified Tollens reaction and
microwave irradiation for Ag NPs synthesis, and quaternized
carboxymethyl chitosan was used as both a reducing and a
stabilizing agent. Nguyen et al. (2013) prepared size-controlled Ag
NPs by autoclaving a mixture of silver-containing glass powder and
glucose, and the results revealed that the Ag NPs were
homogeneously dispersed and embedded in chitosan matrices.
An et al. (2014a) used an inverse-emulsification cross-linking
method for chitosan–Ag NP microparticles preparation. Yadollahi
et al. (2015) used sodium tripolyphosphate as the cross-linker for
one-pot synthesis of chitosan–Ag NP particles.
Numerous papers have proposed techniques for synthesizing
chitosan–silver particles, including the chemical reduction tech-
nique, sol–gel reaction, gamma irradiation synthesis, and green
reduction synthesis (Punitha et al., 2015; Krishna et al., 2015;
Leawhiran et al., 2014). However, fabricating uniform composite
particles in micro size remains challenging (An et al., 2014b).
Droplet microfluidics is an emerging technology for precisely

Table 1
Relationships among the average sizes, deviations, shrink rates, and flow rates.

Flow rate of continuous phase Flow rate of dispersed phase Chitosan emulisions Chitosan particles Shrinking
(mL/min) (mL/h) (%)
Average size SD RSD Average size SD RSD
(mm) (mm) (%) (mm) (mm) (%)
0.6 0.16 370.2 8.8 3.7 326.4 7.8 2.3 11.8
0.12 358.4 9.3 2.4 313.7 7.1 2.1 12.5
0.08 336.3 7.7 2.6 296.8 7.2 1.8 11.7
0.04 315.1 8.3 3.2 276.4 7.4 2.2 12.3
0.02 294.1 8.6 2.9 261.8 7.6 2.5 11
0.5 0.16 435.3 7.8 2.2 381.7 8.1 2.4 12.3
0.12 419.7 8.9 2.1 368.5 7.5 2.6 12.2
0.08 405.6 8.2 2.3 354.7 6.9 1.8 12.5
0.04 391.7 8.8 2.8 346.4 7.9 3.1 11.6
0.02 382.2 8.6 3.1 339.5 8.4 2.4 11.2
0.4 0.16 515.2 7.2 1.5 450.3 7.1 1.8 12.6
0.12 501.5 9.1 1.6 444.2 6.9 1.9 11.4
0.08 486.2 6.7 2.4 423.3 6.7 2.1 12.9
0.04 471.3 8.7 2.4 414.4 7.3 2.2 12.1
0.02 455.1 8.9 2.3 4.5.5 8.1 2.4 10.9
0.3 0.16 629.8 7.6 1.3 557.8 7.6 1.9 11.4
0.12 610.4 8.4 2.5 537.2 7.4 1.8 12
0.08 592.3 8.2 1.9 519 6.2 1.4 12.4
0.04 574.1 8.1 1.6 504.3 8.3 1.7 12.2
0.02 556.2 9.6 2.1 489.7 9.6 2.1 12
 C.-H. Yang et al. / International Journal of Pharmaceutics 510 (2016) 493–500 495

Fig. 2. Optical microscope images of the prepared particles with the flow rate of the dispersed phase fixed at 0.08 mL/h and that of the continuous phase being (A) 0.6 mL/min
and (B) 0.5 mL/min. Scare bars represent 200 mm.

controlling and manipulating fluids to generate monodisperse and 2.3. Synthesis of Ag NP-loaded chitosan particles
uniform droplets in micro size (Yang et al., 2014a,b,c,d, 2009a,b,
2012; Huang et al., 2014, 2011, 2009; Lin et al., 2013, 2012a,b, 2011; The one-step mechanism for synthesizing Ag NP-loaded
Samuel and Klavs, 2010). In this paper, we present a facile and chitosan composite particles involved reducing the Ag NPs and
novel approach for producing uniform Ag NP–chitosan composite solidifying the chitosan particles in emulsions simultaneously
microparticles by using a microfluidic chip. The fabricated (Wang et al., 2015b). Microfluidic synthesis of Ag NP-loaded
composites were characterized, and some antibacterial applica- chitosan particles is depicted in Fig. 1. Briefly, a chitosan solution
tions were subsequently tested. (0.2 g, dissolved in 10 mL of 1%, v/v CH3COOH solution), 5 mL of a
2 M glucose solution, and 5 mL of a 4 mM AgNO3 solution were
2. Materials and methods mixed homogeneously to form a dispersed phase. The concen-
trations of chitosan, glucose, and AgNO3 could be changed for
2.1. Materials subsequent tests. Sunflower seed oil was used as a continuous
phase for sheath flow. The dispersed phase was injected into the
Chitosan (molecular weight: 150000, 1.5% w/v), silver nitrate middle channel from the central inlet, while the continuous phase
(AgNO3), glucose, and sodium hydroxide (NaOH) were purchased was injected from the two side inlets. Syringe pumps (KDSModel
from Sigma–Aldrich (Sigma Chemical Co., St. Louis, MO, USA). 220 Series, Kd Scientific, USA) were used to simultaneously inject
both the dispersed and continuous phases through Teflon tubes
2.2. Microfluidic chip (20 cm) into a microfluidic device. The flow rates of these two
phases changed the shearing force in the cross-junction channel,
The microfluidic chip shown in the inset of Fig. 1 was fabricated and subsequently, monodispersed and uniform emulsions with
as described previously (Yang et al., 2014c). We used a CO2 laser various sizes were obtained. The emulsions dripped from the
machine (LaserPro Venus, Taiwan) for constructing a poly(methyl outlet into a 25 mL 20% NaOH solution for further reduction and
methacrylate) (PMMA) plate with screw orifices, three inlets, one solidification. Composite particles formed after 15 min and were
outlet, and one cross-junction channel. Central and side inlets were retrieved for further washing (twice with 20 mL of water for 5 min)
designed for the dispersed and continuous phases, respectively. and collected (by centrifugation) to remove residual alkali;
The cross-junction channel was designed for flow focus and subsequently, wet Ag NP-loaded chitosan microparticles were
shearing force, and it enabled adjusting the emulsion size by obtained. The collected particles were freeze-dried (EYELA FDU-
changing the flow rates of the dispersed phase or continuous 1100) for 2 days for storing.
phase. The device consisted of three PMMA chips, namely a top
chip (with inlets for sample injection and screw orifices for 2.4. Characterization
binding), a middle chip (with the cross-junction channel and 20
screw orifices), and bottom chip (with an outlet and 20 screw An optical microscope (TE2000U, Nikon, USA) was used to
orifices). The three layers were laminated by using 20 M4 screws observe the emulsions and particles. The average diameter of the
(0.5 mm pitch, 4 mm diameter) and tightened at 1.5–2 Nm. emulsions or particles, expressed as the mean  standard deviation
(SD), was measured in photographs, and approximately 50
Table 2
individual samples were randomly sampled to minimize the
Relationship between the average size of fabricated composite particles and
concentrations of chitosan or NaOH. selection bias. The micromorphology and physical characteristics
of the composite particles were analyzed using a scanning electron
Concentration of chitosan Concentration of NaOH Chitosan particles
microscope (SEM, Hitachi, S-2700, Japan) equipped with an energy
(%) (%)
Average size SD RSD
dispersive X-ray spectrometer (EDS), and a UV–vis spectrometer
(mm) (mm) (%) (Thermo Scientific Spectrascan UV 2700), respectively.
1 10 353.4 6.9 2.2
20 335.1 6.2 2.1 2.5. Cytotoxicity test
30 310.9 7.3 2.6
1.5 10 361.5 6.3 2 The viability of control and treated MCF-7 and NIH 3T3 cells was
20 345.2 6.1 2
measured using a MTT (3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl
30 320.7 6.4 2.3
2 10 377.3 7.0 2.4 tetrazolium bromide) assay. Cells were seeded at a 1 105/well
20 357.3 6.1 2.2 density into a 96-well culture dish plate containing a 100 mL of a
30 327.5 7.4 2.8 culture medium (EMEM Catalog No. 30-2003. or DMEM Catalog
496 C.-H. Yang et al. / International Journal of Pharmaceutics 510 (2016) 493–500
Fig. 3. SEM-EDS photographs of Ag-loaded chitosan microparticles.
No. 30-2002.) and permitted to adhere at 37  C and 5% CO2 for 24 h.
Subsequently, they were treated with various amounts of
composite particles. After 24 h of exposure, 200 mL of an MTT
solution (1 mg/mL) was added for a 4 h reaction with the cells.
Following removal of the medium and the MTT, 100 mL of dimethyl
sulfoxide was added to each well, and the assay plate was read at
OD 595 nm by using a microplate reader (Multiskan Ascent,
Thermo Electron, Finland). The absorbance of the untreated cells in
the control group was considered 100%.
2.6. Antibacterial activity tests
The antibacterial activity of the composites was evaluated by
measuring the inhibition zone against Escherichia coli (E. coli, DH5a
strain) (Annur et al., 2015). E. coli was grown on a sterilized agar
medium containing 100 mL of Luria Broth broth. Composites on
square plastic grids were placed on the solidified agar medium and
incubated at 37  C for 24 h. The inhibition zones were recorded in
four directions for each sample.
3. Results and discussion
3.1. Effects of flow rates on particle size
Table 1 presents the relationships among the average sizes,
deviations, and shrinking rate of emulsions and particles with
various flow rates of the continuous and dispersed phases. The
results revealed that reducing the flow rate of the continuous
phase or increasing the flow rate of the dispersed phase yields
larger emulsions. For example, when the flow rate of the
dispersed phase was fixed at 0.16 mL/h and the flow rate of the
continuous phase was set at 0.6, 0.5, 0.4, or 0.3 mL/min, emulsions
measuring 370.2, 435.3, 515.2, and 629.8 mm were obtained,
respectively. In addition, when the flow rate of the continuous
phase was fixed at 0.6 mL/min and the flow rate of the dispersed
phase was set at 0.2, 0.04, 0.08, 0.12, or 0.16 mL/h, emulsions
measuring 294.1, 315.1, 336.3, 358.4, and 370.2 mm were obtained,
respectively. The microfluidic device can control the emulsion
size to be between 629.8 mm and 294.1 mm, with SDs lower than
10 mm. In addition, all relative standard deviations (RSDs) were
below 4%, indicating that the manufactured emulsions meet the
typical 10% RSD criterion for a monodispersed size distribution.
These uniform size results closely correspond with those
previously reported (Yang et al., 2014a,c, 2009b; Samuel and
Klavs, 2010), evidencing that the proposed droplet microfluidic
device performs effectively (Zhang et al., 2015a). The emulsions
transformed into particles with a shrinking rate of approximately
12% before and after reduction of the Ag NPs and the solidification
of the chitosan particles with a NaOH solution. We observed that
following emulsion shrinkage, the RSDs were still lower than 3%.
This result revealed that the prepared composite particles were
uniform. Two examples of the prepared particles after the
washing process are depicted in Fig. 2. The results reveal that all
 C.-H. Yang et al. / International Journal of Pharmaceutics 510 (2016) 493–500
Fig. 4. MTT assay results for composite particles on (A) NIH 3T3 cells and (B) MCF-7
cells; particle concentrations ranging from 5 to 1000 mg/mL were evaluated. There
were six replicates for each experimental condition.
emulsions and composite particles were spherical and opaque,
with no aggregation.
3.2. Effects of chitosan and NaOH concentrations on the particle size
Generally, the chitosan and NaOH concentrations were set to
1.5% and 20%, respectively. The concentration was increased or
reduced to a general condition to determine the effects of the
chitosan and NaOH concentrations on the particle size. A summary
of these effects is provided in Table 2. The results revealed that the
particle size increased with the chitosan concentration, but
decreased as the NaOH concentration increased when the flow
rates of the dispersed and continuous phases were 0.08 mL/h and
0.5 mL/min, respectively. In addition, the results revealed that all
RSDs were still lower than 3%, evidencing that all prepared
particles were uniform. According to the aforementioned results,
we conclude that the particle size can be adjusted by controlling
the flow rate and the concentrations of chitosan or NaOH. The
particle size varies depending on the chitosan solution concentra-
tion because a concentrated chitosan solution has a higher
viscosity, resulting in a larger size of the emulsion formed in
the microchannel (Yang et al., 2014b, 2009b). In addition, a
497
concentrated NaOH solution has a higher osmotic pressure and
diffusibility, resulting in the anabatic reduction of the Ag NPs and
the solidification of the chitosan particles to form more compact
chitosan particles (Mengmeng et al., 2013).
3.3. Identifying the formation of silver nanoparticles
The SEM with an EDS and the UV–vis spectrometer were used
for measuring the physical characteristics of the composite
particles (Esther et al., 2015; Ştefania et al., 2015). Fig. 3A depicts
the SEM photographs of Ag NP-loaded chitosan composite
microparticles. The results indicated that the surface of the
particle was spheroid, intact, and solid. The elemental mapping at
the microstructural level obtained using the EDS was used to view
the formation and distribution of Ag NPs in the composite
microparticle specimens. Fig. 3B and the inset table present the
amounts of O, C, and Ag atoms in the sequence O > C > Ag. In the
SEM mapping depicted in Figs. 3C–E, the blue, red, and green dots
represent the elemental maps of O, C, and Ag atoms, respectively.
The results confirm that Ag atoms exist and were homogeneously
distributed inside the synthesized composite particles. In addition,
two considerable peaks were observed in the UV–vis spectra of the
composite microparticles. One peak at 275 nm was a characteristic
peak of chitosan, whereas the other peak at 410 nm was a
characteristic peak of nanosized silver particles (Zain et al., 2014;
Mohamed and Sabaa, 2014; Lavorgna et al., 2014; Lin et al., 2013).
According to a literature review, Ag NPs were uniformly dispersed
in the chitosan matrix and captured by the network through amine
( NH2) and hydroxyl ( OH) functional groups (Kandile and Nasr,
2009).
3.4. Biocompatibility tests of the composite particles
Ag NPs have antibacterial properties (Wang et al., 2015a;
Ishihara et al., 2015; Regiel et al., 2013), but their use has been a
cause for concern because they persist in the environment
(Alexander et al., 2015); MCF-7 (cancer cells) and NIH 3T3 (normal
cells) cells were used to determine the biocompatibility of the Ag
NP-loaded chitosan composite microparticles. The results of the
cytotoxicity tests are depicted in Fig. 4. When the concentration of
composite microparticles reached 1000 mg/mL, the cell viability
was >80%, indicating that there was nearly no cell toxicity for both
cell lines. The results proved that Ag NP-loaded chitosan composite
microparticles have high biocompatibility.
3.5. Antibacterial effects
The application of silver noble metal nanoparticles as antibac-
terial agents is an emerging science (Arvizo et al., 2012; Corrêa
et al., 2015; Rizzello and Pompa, 2014). Polymers have provided
higher stability and biocompatibility for Ag NPs (Desireddy et al.,
2013; Ge et al., 2014; Chernousova and Epple, 2013; Wei et al.,
2015; Thakor and Gambhir, 2013). Therefore, Ag NP-loaded
polymer composites can be used as a potential nanomedicine. In
this study, the antibacterial effect against E. coli was investigated
using various concentrations and amounts of composite micro-
particles. In control groups (with no additive), E. coli grew rapidly
within 5 days, as represented by the black bars in Fig. 5A–E. In a
pure chitosan microparticle (without Ag NPs) group, E. coli was
suppressed gradually. For example, the results in Fig. 5A show that
before and after pure chitosan microparticles were treated, the
viability decreased from 160% to 92% on day 2 (DVcontrol chitosan =
Vcontrol Vchitosan = 68%, where DV is the difference in viability,
Vcontrol is the viability of the control, and Vchitosan is the viability of
chitosan), 185% to 82% on day 3 (DVcontrol chitosan = 103%), 248% to
73% on day 4 (DVcontrol chitosan = 175%), and 275% to 65% on day 5
498 C.-H. Yang et al. / International Journal of Pharmaceutics 510 (2016) 493–500

Fig. 5. Comparisons of E. coli viability after treatment with various concentrations, (A) 0.5 mM, (B) 2 mM, (C) 4 mM, (D) 6 mM, and (E) 15 mM and amounts (0.1 g, 0.5 g, and 1 g)
of the Ag NP-loaded chitosan microparticles.

(DVcontrol chitosan = 210%). Several parallel tests in which cells were
treated with various concentrations (0.5 mM–15 mM) and
amounts (0.1 g–1 g) of Ag NP-loaded chitosan composite micro-
particles were conducted. The results revealed that when chitosan
was combined with 0.5 mM Ag NPs, the viability decreased more
significantly than it did when pure chitosan was used. In addition,
when the amounts of Ag NP-loaded chitosan composite
microparticles were increased, the viability decreased. The results
depicted in Fig. 6 indicate that combining chitosan with Ag NPs
yields superior and more effective antibacterial effects than does
chitosan alone; in other words, this finding is consistent with those
of previous studies (Sharma et al., 2009; Rai et al., 2009). In
addition, the inhibition rate was proportional to the amounts of Ag
NPs, indicating that the NPs dose-dependently suppress E. coli
 C.-H. Yang et al. / International Journal of Pharmaceutics 510 (2016) 493–500
Fig. 6. Comparison of E. coli viability after treatment with 0.5 g of Ag chitosan microparticles at various concentrations.
growth (DVcontrol composite: 15 mM group > 6 mM group > 4 mM
group > 2 mM group > 0.5 mM group; DVcontrol composite =
Vcontrol Vcomposite).
4. Conclusion
Compared with other approaches to manufacturing uniform Ag
NP-loaded chitosan composite particles, the proposed microfluidic
method has the following advantages: the facile manufactured
detachable device facilitates easy combination, setup, cleaning,
and reuse; the transparent PMMA chip is suitable for observing
emulsion formation through optical microscopy; the size of
products can be controlled to achieve a narrow size distribution;
and production is automated. In addition, this study is the first to
use a one-step process for the microfluidic synthesis of Ag NP-
loaded chitosan composite microparticles. The results revealed
that various uniform chitosan microparticles impregnated with Ag
NPs were successfully obtained. The composite microparticles
were effective in inhibiting E. coli growth. The results suggest that
the prepared composite microparticles have potential for multiple
applications, particularly as an antimicrobial agent.
Conflicts of interest
The authors declare that they have no competing interests.
Acknowledgments
The authors gratefully acknowledge the support for this study
by I-Shou University and the Ministry of Science and Technology,
Taiwan (102-2632-B-214 -001 -MY3).
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